African trypanosomes are protozoan parasites that cause a fatal disease called sleeping sickness in humans and ngana in domestic livestock. During the past two decades, an estimated 238 million dollars has been spent worldwide on basic research on African trypanosomes. This research has resulted in many advances in our understanding of not only these organisms, but also a number of previously unknown biological phenomena, some of which have been subsequently found to occur in cells of other organisms including mammals. Yet, these substantial research efforts have not led to a better way to manage or control this devastating disease. Recently, progress on the human genome project, and the determination of the yeast genomic sequence, have unambiguously demonstrated the value of using genomic sequences as a foundation for designing future research efforts. Our long term goals is to sequence the genome of Trypanosoma brucei. This proposal represents the first step towards that goal, and we propose the following strategy consisting of two phases. In the first phase, we will generate about 20 Mb of discontinuous single-pass sequence (74 percent of the 27 Mb non-minichromosomal genome). This will be implemented by end-sequencing of 10,000 already-existing clones (currently being used to assemble the physical map of T. brucei isolate TREU 927/4DNA), 5000 BAC clones (currently being made by Sara Melville, Cambridge Univ., UK) and 5000 small insert plasmid clones of randomly- sheared DNA (to be constructed by TIGR). Telomere-proximal sequences containing the telomere-linked vsgs will be identified in clones of the sheared genomic library. The purpose of this first phase is to enhance early gene discovery and to provide markers that will be important for construction of a high-resolution sequence-ready map. During the second phase, 10 Mb of contiguous T. brucei DNA will be sequenced using a chromosome by chromosome approach. This project will provide invaluable information and benefits at many levels, including (1) identification of genes involved in basic functions of the eukaryotic cells, (2) easy, inexpensive and fast cloning of genes encoding proteins being actively studied in laboratories around the world, (3) immediate access to genes and their products from functional/structural studies, (4) prediction of metabolic-pathways on the basis of candidate genes, and (5) identification of parasite-specific gene projects by comparison with other genomes.